Bacterial Mutation Research Articles

Klorin e6’nın Etanol yardımıyla Metisiline Dirençli Stafilokok aureus üzerindeki Fotodinamik Etkisi

Aim: The random use of antimicrobials for years has led to bacterial DNA mutation and a result of that, bacteria have become resistant to antibiotics. Methicillin-resistant Staphylococcus aureus (MRSA) is among these types of resistant bacteria that can easily infect when the immune system of the host is suppressed, and it significantly delays the wound healing. Different treatment methods are being investigated to overcome this problem. Antimicrobial photodynamic therapy is a candidate to become an alternative treatment for the destruction of MRSA. The aim of this study was to investigate the effect of chlorin e6 for the photoinactivation of MRSA and the synergetic role of ethanol in this mechanism.
 Methods: 655 nm laser light and Chlorin e6 as photosensitizer were examined for the photoinactivation of MRSA. Besides, 20% ethanol was used to increase the total antimicrobial efficacy with lower light energy densities and photosensitizer concentrations. The colony counting method was used to determine viable bacterial cells after each application.
 Results: 25 J/cm2 energy density with 20 μM Chlorin e6 and 50 J/cm2 energy density with 10 μM Chlorin e6 showed the highest bactericidal activity. When 20% ethanol was used as an adjuvant, 25 J/cm2 energy dose with 2 μM Chlorin e6 resulted in a better killing effect. 
 Conclusion: Chlorin e6-mediated photodynamic therapy was successful to destroy MRSA and the addition of ethanol provided the opportunity to obtain higher antibacterial activity with lower light intensities and photosensitizer concentrations.

Metabonomics reveals an alleviation of fitness cost in resistant E. coli competing against susceptible E. coli at sub-MIC doxycycline

High concentrations of antibiotics may induce bacterial resistance mutations and further lead to fitness costs by reducing growth of resistant bacteria. However, antibiotic concentrations faced by bacteria are usually low in common environments, which leads to questions about how resistant bacteria with fitness costs regulate metabolism to coexist or compete with susceptible bacteria during sublethal challenge. Our study revealed that a low proportion (< 15%) of resistant bacteria coexisted with susceptible bacteria due to the fitness cost without doxycycline. However, the cost for the resistant strain decreased at a doxycycline concentration of 1 mg/L and even disappeared when the doxycycline concentration was 2 mg/L. Metabonomics analysis revealed that bypass carbon metabolism and biosynthesis of secondary metabolites were the primary metabolic pathways enriching various upregulated metabolites in resistant bacteria without doxycycline. Moreover, the alleviation of fitness cost for resistant bacteria competed with susceptible bacteria at 1 mg/L doxycycline was correlated with the downregulation of the biomarkers pyruvate and pilocarpine. Our study offered new insight into the metabolic mechanisms by which the fitness cost of resistant mutants was reduced at doxycycline concentrations as low as 1 mg/L and identified various potential metabolites to limit the spread of antimicrobial resistance in the environment.

In vivo and in vitro safety evaluation of fermented Citrus sunki peel extract: acute and 90-day repeated oral toxicity studies with genotoxicity assessment

BackgroundCitrus sunki Hort. ex Tanaka peel has been traditionally used as an ingredient in folk medicine due to its therapeutic effects on promotion of splenic health and diuresis as well as relief of gastrointestinal symptoms. Although a growing interest in health-promoting natural products and the development of highly concentrated products have facilitated consumption of C. sunki peel, its safety assessment has not been explored, posing a potential health risk. In this study, we carried out a series of systemic and genetic toxicity tests on fermented C. sunki peel extract (FCPE) to provide the essential information required for safe use in human.MethodsWe conducted acute and 90-day repeated oral toxicity studies in Sprague-Dawley rats to evaluate systemic toxicity, and three genotoxicity assays to measure bacterial mutation reversion, cellular chromosome aberration and in vivo micronucleus formation.ResultsSingle oral administration of FCPE did not cause any clinical signs and lethality in all animals, establishing LD50 to be over 2000 mg/kg BW. Repeated administration of up to 2000 mg/kg BW FCPE for 90 days revealed no test substance-related toxicity as demonstrated in analysis of body weight gain, food/water intake, blood, serum biochemistry, organ weight and histopathology, collectively determining that the no-observable-adverse-effect-level of FCPE is over 2000 mg/kg BW. In addition, we detected no mutagenicity and clastogenicity in FCPE at 5000 μg/plate for the in vitro assays and 2000 mg/kg BW for the in vivo micronucleus test.ConclusionFCPE did not cause systemic and genetic toxicity in our model systems at the tested dose levels. These results suggest a guideline for safe consumption of C. sunki peel in human.

Growth Rate and Antibiotic Sensitivity Effect of Some Natural and Petroleum Based Materials on Staphylococcus aureus

Numerous bacteria expose to different materials every day. Bacterial genome mainly composed of a single double-stranded circular DNA molecule, which can easily undergo changes or mutations upon the exposure to many substances. The aim of this study was to evaluate the mutagenic effect in term of growth rate and antibiotic sensitivity of some natural and petroleum based materials on Staphylococcus aureus. Exposure to each of Bunsen and acetone lower the growth rate of bacterial cells compared to diesel and engine oil that dramatically stimulate their growth. Tobacco based products and the low concentrations of tea and coffee accelerate the growth. The high concentrations of caffeine inhibit the bacterial growth. Wild type bacteria was sensitive to most of used antibiotic and gained resistance to many of them after the exposure to the petroleum products. Similarly, tobacco and tea, which accelerate the growth of cells, make them also completely resistant to the antibiotics that inhibit the synthesis of cell walls. Based on the obtained results, it can be concluded that even natural products can induce bacterial gene mutations such as antibiotic resistance.

The versatility of carbohydrates in antimicrobial applications

AbstractThe worsening situation of global drug resistance is urgently demanding for novel antimicrobial agents. Considerable efforts have been concentrated on developing new antibacterial therapies with new mode of actions, exerting no selective pressure on bacterial mutation, and minimizing toxicity to host cells. In this context, active targeting can greatly contribute to selectivity between pathogens and mammalian cells in which carbohydrates, playing important roles in numerous biological processes, can be employed as targeting ligands or “trojan horse.” This short account has discussed the recent results of carbohydrate‐based antimicrobial agents developed by our group. Other excellent works by other scientists and possible directions in the future are also discussed.

The insect-killing bacterium Photorhabdus luminescens has the lowest mutation rate among bacteria

Mutation is a primary source of genetic variation that is used to power evolution. Many studies, however, have shown that most mutations are deleterious and, as a result, extremely low mutation rates might be beneficial for survival. Using a mutation accumulation experiment, an unbiased method for mutation study, we found an extremely low base-substitution mutation rate of 5.94 × 10–11 per nucleotide site per cell division (95% Poisson confidence intervals: 4.65 × 10–11, 7.48 × 10–11) and indel mutation rate of 8.25 × 10–12 per site per cell division (95% confidence intervals: 3.96 × 10–12, 1.52 × 10–11) in the bacterium Photorhabdus luminescens ATCC29999. The mutations are strongly A/T-biased with a mutation bias of 10.28 in the A/T direction. It has been hypothesized that the ability for selection to lower mutation rates is inversely proportional to the effective population size (drift-barrier hypothesis) and we found that the effective population size of this bacterium is significantly greater than most other bacteria. This finding further decreases the lower-bounds of bacterial mutation rates and provides evidence that extreme levels of replication fidelity can evolve within organisms that maintain large effective population sizes.

Indicators for lack of systemic availability of organic pigments

Experimental data of all 143 organic pigments registered with the European Chemicals Agency, of which 88 were listed in a nanomaterial inventory, was retrieved from the registered substance fact sheets. Availability of the data was 93% for solubility, 82% for bacterial mutagenicity, 79% for acute oral toxicity, 75% for irritation, 59% for skin sensitisation, 36% for repeated dose toxicity and 34% for each clastogenicity and mutagenicity in mammalian cells and 23% for toxicity to reproduction. Pigments mostly had a water and octanol solubility of significantly below 0.1 mg/L, but fourteen were found to be of higher solubility. None were irritating to skin and eyes. Except for the metal salt and the β-naphthol pigments, none of the insoluble pigments showed adverse effects up to limit doses indicating that poor solubility prevents systemic uptake of toxicologically relevant amounts. The few available toxicokinetic data shows absence of metabolism or significant uptake and is in support of this. Occasional effects observed on bacterial mutagenicity and skin sensitisation are attributed to impurities. There is no indication that for organic pigments other particle characteristics such as surface area or morphology have an impact on the investigated toxicological endpoints.

Employing bacterial mutations for the elucidation of photo-Fenton disinfection: Focus on the intracellular and extracellular inactivation mechanisms induced by UVA and H2O2

The bacterial inactivation mechanisms by solar light and the photo-Fenton process is still a matter of debate. In this study, we bring evidence towards the elucidation of the mechanisms that govern photo-Fenton disinfection at near-neutral pH. With the use of porin-deficient and catalase over-producing E. coli strains, in conjunction with measurements of cell wall oxidation and permeability, we are able to i) highlight the role of the aforementioned components in bacterial inactivation and ii) localize the damages in the intracellular domain, despite the addition of the Fenton reagents in the bulk. We report that H2O2 oxidizes cell walls but under light the process is of low significance; UVA initiated an intracellular oxidation process based on excess accumulated H2O2, while the UVA+H2O2 and UVA+H2O2+Fe2+ processes have the same effect with light, albeit enhanced, as shown by malondialdehyde (MDA) production and ONPG hydrolysis rates. Finally, compared to the UVA-assisted photo-Fenton process, its solar counterpart is enhanced by the direct UVB effects on bacterial DNA. In conclusion, we have sufficient evidence to postulate that the photo-Fenton process is intracellular and propose the pathways that form the integrated bacterial inactivation mechanism by photo-Fenton.

New Delhi metallo-β-lactamase-1 inhibitors for combating antibiotic drug resistance: recent developments

The most common mechanism of the resistance to β-lactam antibiotics is the expIdentification of cisplatin and palladiuression of β-lactamases, which can hydrolyze the β-lactam moiety and deactivate these antimicrobials. The worldwide prevalence of New Delhi metallo-β-lactamase-1 (NDM-1), also known as carbapenemase has created distress among clinicians. NDM-1 is a type of metallo β-lactamase (type of β-lactamases which require metal ions for their catalytic action) produced in pathogens carrying blaNDM-1 gene. These NDM-1 producing pathogens are resistant to all β-lactam antibiotics including carbapenems and are greatest threat to public health as they can easily extend via horizontal gene transfer. Various NDM-1 variants have been evolved over time and it may further evolve in the future due to several factors such as bacterial gene mutation and overuse of antibiotics. In the past 10 years, various NDM-1 inhibitors have been reported showing diverse chemical structure. In spite of a great development in terms of structural and mechanistic information, the design of a potent inhibitor of NDM-1 to be approved clinically remains challenging, this could be due to structural complexity of the enzyme that limits the development of clinically useful NDM-1 inhibitors. Along with designing of novel and effective NDM-1 inhibitors, measures should be taken for controlling multidrug resistance. Currently, there is non-availability of NDM-1 inhibitors clinically therefore clinicians are facing a huge challenge in treating infections due to multidrug-resistant bacteria. This review article has been planned to discuss about antibiotic resistance, NDM-1, mechanism of antibiotic resistance by NDM-1, and recent updates in the development of NDM-1 inhibitors as well as mechanism of action of NDM-1 inhibitors.

Effect of Short-Term Antimicrobial Therapy on the Tolerance and Antibiotic Resistance of Multidrug-Resistant Staphylococcus capitis.

BackgroundBacteria undergo adaptive mutation in the host. However, the specific effect of antimicrobial use on bacterial evolution and genome mutations related to bacterial survival within a patient is unclear.Materials and MethodsThree S. capitis strains were sequentially isolated from cerebrospinal fluid of a clinical inpatient. Antimicrobial susceptibility, growth rate, biofilm formation and whole blood survival of these strains were measured. Relative fitness was calculated. The virulence was examined in the Galleria mellonella model. Whole-genome sequencing and in silico analysis were performed to explore the genetic mechanisms of the changes in antimicrobial resistance phenotype. Hypothetical proteins are cloned, expressed and characterized by detection the susceptibility to gentamycin.ResultsThe first isolate was susceptible to rifampin (MIC=0.25 μg/mL), resistant to gentamicin (MIC=16 μg/mL), while the later two isolates were resistant to rifampin (MIC >64 μg/mL), susceptible to gentamicin (MIC=4 μg/mL). For the latter two strains, compared to the first, frameshift mutation in a hypothetical protein encoding gene and base substitutions (in genes saeR, moaA and rpoB) were discovered. The mutation of rpoB gene caused rifampicin resistance. Mutations in saeR, moaA and hypothetical gene are associated with changes in other biological traits. Amino acid sequence-based structure and function identification of the hypothetical protein indicated that a mutation in the encoding gene might be associated with altered aminoglycoside susceptibility. Growth curve showed that the later two isolates grew faster than the first isolate with a positive fitness advantage of 13.5%, and 14.8%, accordingly. Biofilm form ability and whole blood survival of the derivative mutants were also enhanced. No significant differences of virulence in the G. mellonella model were observed.ConclusionWe report here for the first time that short-term clinical antibiotic use was associated with resistance mutations, collateral sensitivity, and positive in vivo fitness advantages to S. capitis during infection.

Characterization of colibactin-associated mutational signature in an Asian oral squamous cell carcinoma and in other mucosal tumor types

Mutational signatures can reveal the history of mutagenic processes that cells were exposed to before and during tumorigenesis. We expect that as-yet-undiscovered mutational processes will shed further light on mutagenesis leading to carcinogenesis. With this in mind, we analyzed the mutational spectra of 36 Asian oral squamous cell carcinomas. The mutational spectra of two samples from patients who presented with oral bacterial infections showed novel mutational signatures. One of these novel signatures, SBS_AnT, is characterized by a preponderance of thymine mutations, strong transcriptional strand bias, and enrichment for adenines in the 4 bp 5′ of mutation sites. The mutational signature described in this manuscript was shown to be caused by colibactin, a bacterial mutagen produced by E. coli carrying the pks-island. Examination of publicly available sequencing data revealed SBS_AnT in 25 tumors from several mucosal tissue types, expanding the list of tissues in which this mutational signature is observed.

Mycobacterium tuberculosis clinical isolates carry mutational signatures of host immune environments.

Mycobacterium tuberculosis (Mtb) infection results in a spectrum of clinical and histopathologic manifestations. It has been proposed that the environmental and immune pressures associated with different contexts of infection have different consequences for the associated bacterial populations, affecting drug susceptibility and the emergence of resistance. However, there is little concrete evidence for this model. We prospectively collected sputum samples from 18 newly diagnosed and treatment-naïve patients with tuberculosis and sequenced 795 colony-derived Mtb isolates. Mutant accumulation rates varied considerably between different bacilli isolated from the same individual, and where high rates of mutation were observed, the mutational spectrum was consistent with reactive oxygen species-induced mutagenesis. Elevated bacterial mutation rates were identified in isolates from HIV-negative but not HIV-positive individuals, suggesting that they were immune-driven. These results support the model that mutagenesis of Mtb in vivo is modulated by the host environment, which could drive the emergence of variants associated with drug resistance in a host-dependent manner.

Reduction in CYP1A1 and 2B2 activity at low oxygen tension

The Cytochrome P450 (CYP) enzyme family comprises a wide array of monooxygenases involved in the oxidation of endobiotic and xenobiotic molecules. The active site of a CYP enzyme contains an iron protoporphyrin center coordinated to a cysteine thiolate, and then, molecular oxygen is associated with the iron to be converted into dioxygen complex plus substrate. Reduction by CYP reductase expedites hydroxylation of the compound. In this oxidation reaction, insufficient oxygen molecules would affect enzyme catalysis. Nevertheless, biochemical data about CYP kinetics at low oxygen concentrations are not available. In this work, we present the results on the variation in rat liver microsomal CYP Vmax app and Km app under normal and hypoxic conditions. Using alkoxyresorufin molecules as substrates, the Vmax/Km ratios for resorufin production decreased from 426 to 393 for CYP1A1 and from 343 to 202 for CYP2B1 at a low oxygen concentration (4.1 ppm) compared to the ratios observed at a normal oxygen concentration (6.5 ppm). Additionally, the bacterial mutagenicity of 2-aminoanthracene and cyclophosphamide, decreased by 32% and 42%, respectively, at low oxygen concentrations. These results support the hypothesis that low oxygen availability is implicated in the low efficiency of substrate oxidation by CYP.

Evaluation of 2-methoxy-4-nitroaniline (MNA) in hypersensitivity, 14-day subacute, reproductive, and genotoxicity studies

2-Methoxy-4-nitroaniline (MNA), an intermediate in the synthesis of azo dyes used in textiles and paints, is structurally similar to carcinogenic anilines. Human exposure occurs primarily in the occupational setting through handling of dye dust, and through use and disposal of MNA-containing products. MNA has been reported to induce contact hypersensitivity in a human, myocardial necrosis in rats, and bacterial mutagenicity. This study assessed the subacute toxicity, genotoxicity, contact hypersensitivity, and reproductive toxicity of MNA in rodents in an effort to more fully characterize its toxicological profile. B6C3F1/N mice were exposed to 0, 650, 1250, 2500, 5000, or 10,000 ppm MNA by dosed feed for 14-days to evaluate subacute toxicity and histopathological endpoints. In female mice, decreased body weight (13.5 %) and absolute kidney weight (14.8 %), compared to control, were observed at 10,000 ppm MNA; increased relative liver weight (10–12 %), compared to control, occurred at 5,000–10,000 ppm MNA. In male mice, absolute (15 %) and relative liver weights (9–13 %) were increased at 2,500−5,000 ppm and 1250–10,000 ppm MNA, compared to control, respectively. In both sexes of mice, minimal elevations of hemosiderin pigmentation (a breakdown product of erythrocytes), relative to control, were observed in the liver (10,000 ppm); minimal to moderate elevations of hemosiderin pigmentation (5,000–10,000 ppm) and minimal increases in hematopoietic cell proliferation occurred in the spleen (≥ 1250 ppm). In a reproductive toxicity study, timed-mated female Harlan Sprague Dawley rats were exposed to 0–10,000 ppm MNA by dosed feed from gestation day 6 through postnatal day (PND) 21. Decreases in mean litter weights were observed at 5000 ppm MNA, compared to control, beginning at PND1. To evaluate potential contact hypersensitivity, MNA (2.5–50 %, in dimethylformamide) was applied to the dorsa of both ears of female Balb/c mice once daily for three days. The increase observed in lymph node cell proliferation (10–50 % increase in thymidine uptake compared to control) did not reproducibly achieve the Sensitization Index (SI) 3 level, and there was no ear swelling evident following sensitization with 10–50 % MNA and challenge with 25 % MNA in the mouse ear swelling test. In bacterial mutagenicity assays, MNA (250−1000 μg/plate) induced significant increases, compared to control, in mutant colonies with and without metabolic activation enzymes in Salmonella typhimurium strains TA100 and TA98. These data indicate that MNA is genotoxic, and may induce erythrocyte damage and reactive phagocytosis by macrophages in the liver and spleen.

Investigating DNA adduct formation by flavor chemicals and tobacco byproducts in electronic nicotine delivery system (ENDS) using in silico approaches

The presence of flavors is one of the commonly cited reasons for use of e-cigarettes by youth; however, the potential harms from inhaling these chemicals and byproducts have not been extensively studied. One mechanism of interest is DNA adduct formation, which may lead to carcinogenesis. We identified two chemical classes of flavors found in tobacco products and byproducts, alkenylbenzenes and aldehydes, documented to form DNA adducts. Using in silico toxicology approaches, we identified structural analogs to these chemicals without DNA adduct information. We conducted a structural similarity analysis and also generated in silico model predictions of these chemicals for genotoxicity, mutagenicity, carcinogenicity, and skin sensitization. The empirical and in silico data were compared, and we identified strengths and limitations of these models. Good concordance (80–100%) was observed between DNA adduct formation and models predicting mammalian mutagenicity (mouse lymphoma sassy L5178Y) and skin sensitization for both chemical classes. On the other hand, different prediction profiles were observed for the two chemical classes for the modeled endpoints, unscheduled DNA synthesis and bacterial mutagenicity. These results are likely due to the different mode of action between the two chemical classes, as aldehydes are direct acting agents, while alkenylbenzenes require bioactivation to form electrophilic intermediates, which form DNA adducts. The results of this study suggest that an in silico prediction for the mouse lymphoma assay L5178Y, may serve as a surrogate endpoint to help predict DNA adduct formation for chemicals found in tobacco products such as flavors and byproducts.

Commensal Bacteria Modulate Immunoglobulin A Binding in Response to Host Nutrition

Immunoglobulin (Ig) A controls host-microbial homeostasis in the gut. IgA recognition of beneficial bacteria is decreased in acutely undernourished children, but the factors driving these changes in IgA targeting are unknown. Child undernutrition is a global health challenge that is exacerbated by poor sanitation and intestinal inflammation. To understand how nutrition impacts immune-microbe interactions, we used a mouse model of undernutrition with or without fecal-oral exposure and assessed IgA-bacterial targeting from weaning to adulthood. In contrast to healthy control mice, undernourished mice fail to develop IgA recognition of intestinal Lactobacillus. Glycan-mediated interactions between Lactobacillus and host antibodies are lost in undernourished mice due to rapid bacterial adaptation. Lactobacillus adaptations occur in direct response to nutritional pressure, independently of host IgA, and are associated with reduced mucosal colonization and with bacterial mutations in carbohydrate processing genes. Together these data indicate that diet-driven bacterial adaptations shape IgA recognition in the gut.

Identification of a transcriptomic signature of food-relevant genotoxins in human HepaRG hepatocarcinoma cells

The conventional approach for testing the genotoxic potential of chemicals in vitro includes a battery of bacterial and mammalian mutagenicity tests. Toxicogenomics analyses may provide information about DNA-damaging properties of test compounds but are not routinely used for identification of a genotoxic potential. In this study, metabolically active human HepaRG hepatocarcinoma cells were exposed to five food-relevant genotoxic carcinogens. Transcriptomic responses were analyzed using RNA sequencing technology and validated by real-time polymerase chain reaction. Biostatistical approaches revealed a characteristic transcript signature of 37 differentially expressed genes, which were commonly regulated by the test chemicals. Specificity of the transcript signature was confirmed by using non-genotoxic carcinogens as comparators. Pathway analyses showed that the obtained transcript signature was closely related to DNA damage response and p53 activation. In conclusion, we have established a characteristic transcript marker pattern to monitor genotoxicity in human HepaRG cells, and to distinguish genotoxic from non-genotoxic carcinogens. Our analyses underline that a common response related to DNA damages response, cell cycle alterations and cell death is initiated in HepaRG cells upon exposure to genotoxic compounds and allows for the identification of a common transcriptomic signature for genotoxic stress.

Increased Quinolone-Resistant Mutations of gyrA and parC Genes after Pouchitis Treatment with Ciprofloxacin

Background: Oral antibiotics, such as ciprofloxacin (CFX), are widely used for the treatment of acute and chronic pouchitis. Most bacterial mutations that confer quinolone resistance are at Ser-83 and Asp-87 in the gyrA gene and Ser-80 and Glu-84 in the parC gene. Methods: We obtained 51 stool samples from 43 patients who were diagnosed with ulcerative colitis and underwent ileal pouch-anal anastomosis. Patients were divided into 2 groups: 13 patients with CFX treatment of pouchitis and 30 patients without pouchitis. After extraction of fecal DNA, the amount of Escherichia coli 16S rRNA, gyrA, and parC gene DNA were measured using real-time polymerase chain reaction (PCR). Possible mutations at gyrA 83 and 87 and at parC 80 and 84 were investigated by PCR cloning and sequencing, and mutation rates were quantified by rapid PCR-restriction fragment length polymorphism. Results: Samples from both CFX-treated and -untreated patients had comparable levels of gyrA and parC gene DNA. Nucleic acid and amino acid mutations were identified at gyrA 83 and 87, and at parC 80 and 84. We successfully quantified mutation rates at gyrA 83 and 87, and at parC 84, all of which were significantly higher in samples from CFX-treated patients (70, 84, and 38%) than from CFX-untreated patients (13, 11, and 5%). Conclusion: E. coli in patient pouches may have mutations in their gyrA and parC genes that produce CFX resistance. Mutation rates of these genes were significantly higher in samples from CFX-treated patients. This study contributes to understanding the decrease and loss of CFX effectiveness against pouchitis.

Impact of anticancer chemotherapy on the extension of beta-lactamase spectrum: an example with KPC-type carbapenemase activity towards ceftazidime-avibactam

Through their action on DNA replication, anticancer chemotherapies could increase the basal mutation rate in bacteria and increase the risk of selecting antibiotic resistant mutants. We investigated the impact of several drugs on a beta-lactamase model using KPC-type carbapenemase-producing Enterobacteriaceae. We studied the impact of anticancer chemotherapies used in pediatric hematologic malignancies on 7 clinical isolates of Enterobacteriaceae producing KPC-type carbapenemases. We compared the mutation rates from cultures with/without chemotherapy on ceftazidime-avibactam, rifampicin and ceftazidime-avibactam combined with meropenem media. Mechanisms of ceftazidime-avibactam resistance were explored on a subset of mutants. After exposure to some cytotoxic molecules, the bacterial mutation rates leading to ceftazidime-avibactam and to rifampicin resistance increased up to 104-fold while we observed no emergence of resistant mutants (frequency of <10−10) on a meropenem combined with ceftazidime-avibactam media. Compared to the parental strains, an increased susceptibility to meropenem was observed in the ceftazidime-avibactam resistant mutants. The blaKPC genes of ceftazidime-avibactam mutants harbored either mutations, deletions or insertions, especially in the region encoding the Ω-loop of the KPC-type carbapenemase. Anticancer chemotherapy can increase the mutation rates of bacteria accelerating the extension of KPC-type carbapenemases towards ceftazidime-avibactam, one of the last resort antimicrobial chemotherapy.

Preclinical safety assessment of pathogen reduced red blood cells treated with amustaline and glutathione.

ABSTRACTBACKGROUNDThe nucleic acid targeted pathogen reduction (PR) system utilizing amustaline (S‐303) and glutathione (GSH) is designed to inactivate blood‐borne pathogens and leukocytes in red blood cell concentrates (PR‐RBCC). Inactivation is attained after amustaline intercalates and forms covalent nucleic acid adducts preventing replication, transcription, and translation. After pathogen inactivation, amustaline spontaneously hydrolyzes to S‐300, the primary negatively charged reaction product; amustaline is below quantifiable levels in PR‐RBCC. GSH quenches free unreacted amustaline.STUDY DESIGN AND METHODSThe genotoxic and carcinogenic potential of PR‐RBCC, the reaction by‐products, and S‐300 were assessed in accordance with the International Conference on Harmonization (ICH) guidelines and performed in compliance with the Food and Drug Administration (FDA) good laboratory practice standards, 21 CFR Part 58. in vitro bacterial reverse mutagenicity and chromosomal aberration assays were performed with and without exogenous S9 metabolic activation, and in in vivo clastogenicity and carcinogenic assays using validated murine models.RESULTSPR‐RBCCs were not genotoxic in vitro and in vivo and were non‐carcinogenic in p53+/− transgenic mice transfused over 26 weeks. Estimated safety margins for human exposure ranged from >90 to >36 fold for 2 to 5 PR‐RBCCs per day, respectively. PR‐RBCCs and S‐300 did not induce chromosome aberration in the in vivo murine bone marrow micronucleus assay at systemically toxic doses.CONCLUSIONSPR‐RBCCs did not demonstrate genotoxicity in vitro or in vivo and were not carcinogenic in vivo. These studies support the safety of PR‐RBCCs and suggest that there is no measurable genotoxic hazard associated with transfusion of PR‐RBCCs.